The vitamin D receptor (VDR) is a transcription factor that mediates the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the nucleus of target cells. The receptor binds to target genes and regulates their transcriptional output. While the VDR gene is expressed and regulated in numerous cell types, almost nothing is known of the tissue-specific determinants of its expression that may provide unique therapeutic opportunity. Our recent studies have defined the boundaries and specific regulatory features of the mouse and human genes and shown their utility as transgenes to rescue the VDR null mouse. Therefore, three aims are proposed. Aim 1: To identify intestinal, renal and bone-specific enhancers in mouse and human VDR genes, to identify key regulatory factors and to examine enhancer structure and function. Based upon our approach in bone cells using ChIP-chip/ChIP-seq analyses, we will extend our search for regulatory determinants in mouse and human VDR genes in intestinal and renal tissues and in tissue-related cell lines. We will identify enhancers using signature-defining chromatin marks such as H3K4me1, H3K9ac and H4K16ac, explore transcription factor interactions at these regions and assess enhancer structure and function. Aim 2: To define the roles of mouse and human VDR gene enhancers in tissue-specific and regulated expression of VDR gene in vivo and to assess the consequence of enhancer deletion on biologic phenotype. Mouse and human DNA transgene segments have been identified that mimic endogenous gene regulatory capability and rescue the aberrant phenotype of VDR deficient mice. We will explore individual enhancer contributions to tissue-specific and regulated expression of VDR transgenes and assess their ability to rescue the VDR null mouse phenotype. Functional and targeting properties will also be assessed in vivo. Features unique to the human VDR BAC transgene will be similarly examined, including the role of an alternative promoter located upstream. Aim 3: To examine the biologic consequence of selected human VDR coding mutations in the humanized VDR null mouse in vivo. A human VDR BAC transgene rescues the VDR null mouse. We will extend this finding to examine the effect of selective mutations inserted into the coding region of the human transgene on VDR function in vivo and on the phenotype in the VDR null mouse. Several mouse models of hereditary 1,25(OH)2D3 resistant rickets will be created and evaluated. We will also examine the consequences of preventing phosphorylation of the human VDR at residue S208 and perhaps other sites as well. These novel studies will provide new insight into mechanisms that govern VDR gene expression and function and may provide unique therapeutic opportunity. PUBLIC HEALTH RELEVANCE: Vitamin D plays significant roles both in the maintenance of mineral homeostasis and in the control of cellular growth and function. The studies herein seek to enhance our understanding of the mechanisms that underlie vitamin D action through its receptor in bone and other cells such that better and more selective medicines can be created.